Identification and characterization of a novel alpha-amylase from maize endosperm

ABSTRACT

SHE, a Starch Hydrolytic Enzyme active in maize endosperm ( Zea mays ), and the cDNA sequence encoding SHE are disclosed. The specificity of native, purified SHE is similar, in general terms, to previously known alpha-amylases. However, the activity of SHE toward amylopectin results in hydrolysis products that are distinctly different from those of other alpha-amylases. SHE, and its homologous equivalents in other plants such as rice,  Arabidopsis , apple and potato, can be used in starch processing for generating different, e.g., larger sized, alpha-limit dextrins for industrial use, as compared to those generated by previously known alpha-amylases or other starch hydrolytic enzymes. In addition, modification of the expression of this enzyme in transgenic maize plants or in other transgenic organisms (including bacteria, yeast, and other plant species) can be useful for the generation of novel starch forms or altered starch metabolism.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 10/952,551 entitled IDENTIFICATION AND CHARACTERIZATION OF ANOVEL ALPHA-AMYLASE FROM MAIZE ENDOSPERM filed Sep. 27, 2004 and claimspriority benefit of U.S. Provisional Application No. 60/505,995, filedSep. 25, 2003, entitled IDENTIFICATION AND CHARACTERIZATION OF A NOVELALPHA-AMYLASE FROM MAIZE ENDOSPERM, the whole of which are herebyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

BACKGROUND OF THE INVENTION

Starch is the major storage carbohydrate in higher plants. Thebiochemical mechanisms of starch biosynthesis and starch utilization areof interest for understanding fundamental aspects of plant physiologyand also for their potential utility in manipulating the starch pathwayfor practical purposes. Not only is starch a critical primary source ofdietary carbohydrates, but it is also used extensively for variousindustrial purposes ranging from formation of packaging materials toethanol production. Despite its wide availability in nature and its manyindustrial applications, the mechanisms by which starch is formed anddegraded in plant endosperm tissue are not well understood.

Starch consists essentially of a mixture of the homopolysaccharidesamylose and amylopectin [11, 24]. Amylose is a linear chain of glucosylunits joined by alpha-1,4 glycosidic bonds and normally constitutesabout 256 of the total endosperm starch in maize (Zea mays). Amylopectincomprises many linear chains of glucosyl monomers joined by alpha-1,4linkages and constitutes approximately 75 W of the starch. The chains ofamylopectin are joined to each other by alpha-1,6 glycosidic bonds,often referred to as branch linkages. In amylopectin, the organizedpositioning of branch linkages enables periodic clustering of the linearchains [9, 15]. This permits tight and efficient packaging of glucoseunits, and confers crystallinity to the granule. The functionalproperties of starch relate directly to this architectural organizationof linear chains and branch linkages in amylopectin [23].

The organization of amylopectin and amylose into higher order structuresthat lead to granule formation renders starch resistant to degradation.However, starch granules can be completely degraded by a combination ofphosphorolysis and hydrolysis when glucose supply is required [2]. As nosingle enzyme has been shown to completely convert starch to simplesugars, multiple enzymes most likely are involved. Starch debranchingenzymes and disproportionating enzymes are potential degradativeenzymes, as are the alpha-1,4 linkage-specific hydrolases of thealpha-amylase and beta-amylase classes. Genetic evidence for involvementof an alpha-amylase in starch degradation comes from the sex4 mutant ofArabidopsis, which lacks such an enzyme and accumulates abnormally highlevels of starch in leaves [34]. Another enzyme that likely participatesin starch degradation is phosphorylase, which inserts phosphoryl groupsfrom inorganic pyrophosphate into the alpha-1,4 glucoside bond,releasing glucose-1-phosphate.

Classification of starch degrading enzymes is made according to theirbehavior: endo- versus exo-mode of attack, inversion versus retention ofanomeric configuration of the substrate, preference for length of theglucosyl chain, preference for the nature of the glucosyl bond, andhydrolytic versus glucosyl-transfer activity [31]. Alpha-amylases areendo-acting hydrolytic enzymes that hydrolyze internal alpha-1,4linkages in alpha D-glucan polymers, such as amylopectin and amylosemolecules. Alpha-amylases are widely distributed in nature, and areproduced by plants, animals, and microorganisms [28]. Those fromdifferent sources are known to have different substrate specificities,acting preferentially on glucan chains of different lengths. Thissubstrate specificity is dependent on the configuration of the activesite of the enzyme and results in characteristic products that areformed according to the enzyme source [28]. For example, salivary glandand pancreatic alpha-amylases immediately produce low molecular weightproducts such as maltose and maltotriose by “multiple attack” on thesubstrate [27], and barley alpha-amylase primarily produces maltose,maltohexaose, and maltoheptaose without multiple attack [1,9]. Becausealpha-amylases can hydrolyze linkages only so close to a branch point(generated by an alpha-1,6 linkage), activity halts when this physicallimitation occurs. When hydrolytic activity of the alpha-amylase reachesthis limit, the resulting product is termed a “limit dextrin”. Toachieve further hydrolysis of the limit dextrin, other enzymes must beemployed, such as exo-cleaving beta-amylases or debranching enzymes. Allof the alpha-amylase activities that have been described to datehydrolyze alpha D-glucans to maltose, maltotriose, or other smallmalto-oligosaccharides plus alpha-limit dextrins of various sizes [19,28, 31].

Hydrolysis of starch with alpha-amylases from bacteria or fungi isroutinely used by some starch industries as a first step in the processof the complete degradation of starch to glucose (this step is termed“saccharification”). The hydrolysis of starch to glucose is preliminaryto the manufacture of conversion products such as high fructose cornsyrup or fuel ethanol [18]. The goal of other starch processingindustries is the incomplete hydrolysis of starch by various degradativeenzymes, including alpha-amylases, to generate limit dextrins (termed“maltodextrins”) in a range of sizes that are used for a variety ofindustrial purposes. For example, maltodextrins are used in food andpharmaceutical manufacturing as thickening agents, cryoprotectants andbinders. They can also be further processed or chemically modified foruse as viscosity or hygroscopicity or dissolving agents [13]. Differentlimit dextrin products are typically produced by varying the combinationof enzymes used for the starch digestion, or by varying the digestionconditions. An important industrial goal is the low-energy production ofspecific starch hydrolysates containing few by-products [18].

In plants, alpha-amylases are believed to be involved in the hydrolysisof transient starch in the leaves, which occurs during the dark cycle ofthe plant, and in the hydrolysis of storage starch that accumulates inseeds or tubers, which occurs during seed germination or tubersprouting. The first complete sequence of a plant genome, that of theArabidopsis genome, reveals that three alpha-amylase genes are presentin this plant species [14, 16]. Two are genes that encode predictedpolypeptides of approximately 50-60 kilodaltons (kD), and one is a genethat encodes a larger form predicted to have a molecular mass ofapproximately 100 kD (Genbank Accession No. NM_(—)105651). Sequencing ofthe rice genome reveals the presence of one homolog of the Arabidopsisgene that encodes the large alpha amylase [12]. This rice gene is alsopredicted to encode a polypeptide of approximately 100 kD (GenbankAccession No. AP003408). In addition, the rice genome contains severalgenes that code for smaller sized (50-60 kD) alpha-amylases. All of theplant 50-60 kD alpha-amylases are similar in size to those frombacteria, yeast, and mammals that are used commercially. Activities ofthe 50-60 kD alpha-amylase enzymes from plants also are similar to thoseof bacterial, fungal, and mammalian alpha-amylase enzymes, in that theyresult in starch hydrolysis products consisting of maltose, maltotriose,or small oligosaccharides plus alpha limit dextrins. The activities ofthe 100 kD plant alpha-amylases and the nature of their starchhydrolysis products have not been characterized to date.

Alignment of the amino acid residues of all predicted alpha-amylases(both large and small) reveals they are highly similar in theirC-terminal regions, which are believed to contain the catalytic domainof the protein [17]. The two 100 kD alpha-amylases from Arabidopsis andrice also have considerable amino acid sequence similarity, with 37%sequence identity in their N-terminal regions and 59% amino acididentity overall. The N-termini of the Arabidopsis and rice 100 kDalpha-amylases also have two small regions of similarity with anotherprotein from Arabidopsis that has been termed the R1-protein, theproduct of the sex1 gene [26, 35]. Mutations in the sex1 gene result inexcess starch accumulation, suggesting that a functional R1-protein isrequired for starch degradation. This suggests the larger 100 kDalpha-amylases from plants comprise a distinct isoform class ofalpha-amylase enzymes. Further investigation into the role of the largealpha-amylase in starch metabolism, particularly in an agronomicallyimportant plant such as maize, is desirable.

BRIEF SUMMARY OF THE INVENTION

The invention is based on the discovery of a novel starch hydrolyticactivity (called Starch Hydrolytic Enzyme, or SHE) in developing maizekernels. The specificity of native, purified SHE is similar, in generalterms, to previously known alpha-amylases since both activities are ableto hydrolyze starch, amylopectin, amylose, and beta-limit dextrin butare not able to hydrolyze the branched polymer pullulan. However, theactivity of SHE toward amylopectin results in hydrolysis products thatare distinctly different from those of other alpha-amylases.Specifically, they are of the same approximate molecular mass asbeta-limit dextrins and do not include maltose ormalto-oligosaccharides. This unique activity suggests that the novelmaize alpha-amylase is an endo-hydrolytic enzyme that specificallycleaves long amylopectin chains (B₂ or B₃ chains) that extend betweenunit clusters in the molecule. In contrast, conventional maize amylases,in addition, clip off the smaller side chains of amylopectin. The newenzyme according to the invention, SHE, and its homologous equivalentsin other plants such as rice, Arabidopsis, apple and potato, will havevalue in starch processing for generating different, and perhaps largersized, alpha-limit dextrins for industrial use, as compared to thosegenerated by previously known alpha-amylases or other starch hydrolyticenzymes. In addition, modification of the expression of this enzyme intransgenic maize plants or in other transgenic organisms (includingbacteria, yeast, and other plant species) can be useful for thegeneration of novel starch forms or altered starch metabolism.

The cDNA encoding the new enzyme according to the invention, SHE, hasalso been isolated and sequenced. cDNA sequences encoding SHE orportions thereof can be incorporated into replicable expression vectorsand the vectors transfected into an appropriate host (e.g., bacterial,yeast, eucaryotic cell culture). Alternatively, genomic DNA fragmentsencoding SHE can be utilized in situ. The SHE protein, in eithernaturally occurring or recombinant form, can be used in the starchprocessing industry or in other industries that employ starch for anypurpose. The protein, or fragments thereof, also can be employed as animmunogen in order to raise antibodies against SHE.

Thus, the invention generally features a Starch Hydrolytic Enzyme, SHE,or portions thereof; nucleic acid isolates encoding SHE or portionsthereof; methods of producing SHE or portions thereof; cells transformedwith a recombinant vector containing a SHE-encoding alpha-amylase3(Amy3) gene; antibodies to SHE or fragments thereof and methods toproduce such antibodies; transgenic plants containing a SHE gene andmethods to produce such transgenic plants; and methods of using aprotein having SHE hydrolytic activity for starch degradation.

The invention also features a nucleic acid isolate able to hybridizeunder stringent conditions to the complement of a nucleic acid sequenceencoding SHE, and the protein or polypeptide fragment, e.g., immunogenicfragment, thereof encoded by the nucleic acid isolate. The invention,furthermore, features a recombinant expression vector comprising anucleic acid isolate able to hybridize under stringent conditions to thecomplement of a sequence encoding SHE, cells transformed with therecombinant expression vector, and methods of expressing the SHE proteinor polypeptide fragment encoded within the recombinant expressionvector.

Also featured is a method of producing the SHE protein, or polypeptidefragment thereof, comprising transforming a host cell with a nucleicacid able to hybridize under stringent conditions to a nucleic acidsequence encoding the SHE protein and linked to a nucleic acid sequenceunder the control of an inducible promotor, and inducing the cell toproduce a fusion protein comprising the SHE protein, or polypeptidefragment thereof. The invention also features a SHE fusion protein,methods of producing antibodies to a SHE fusion protein and antibodiesproduced by such method.

As used herein, the terms “isolated” or “purified” refer to a nucleicacid or protein sequence that has been separated or isolated from theenvironment in which it was prepared or in which it naturally occurs.Such nucleic acid or protein sequences may be in the form of chimerichybrids or fusions, useful for combining the function of the nucleicacid or protein sequences of the invention with other species and alsoinclude recombinant forms. The term “determinant” as used hereinincludes the site on an antigen at which a given antibody moleculebinds. The term “immunogenic fragment” refers to a fragment of SHEprotein that reacts with antibodies specific for a determinant of SHE.

The SHE protein can be used as an alternative hydrolase, along withbacterial and fungal starch hydrolases and debranching enzymes, forindustrial starch processing applications. SHE-encoding cDNA (Amy3),SHE-encoding genomic DNA (Amy3), or portions thereof may be utilized asmarkers for the identification of specific corn varieties, and for thedevelopment of corn varieties with starch properties tailored forspecific industrial applications. Amy3 cDNA or genomic DNA fragments canbe used to produce these proteins or peptide fragments or as probes toidentify nucleic acid molecules encoding related proteins orpolypeptides (e.g., homologous polypeptides from related species andheterologous molecules from the same species). Assays for SHE function,production or expression by cells are made possible by the developmentof antibodies reactive with the SHE protein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims, taken in conjunction with the accompanying drawings in which:

FIG. 1 is a gel showing two-dimensional separation ofstarch-metabolizing enzymes from developing maize endosperm;

FIG. 2 is a flow chart showing a purification scheme for purifying theStarch Hydrolytic Enzyme (SHE) according to the invention;

FIG. 3 is a gel analysis of hydrolysis results showing the specificityof SHE toward different polysaccharides;

FIGS. 4A and 4B are graphs showing FACE analysis of hydrolysis productsof amylopectin incubated with SHE (FIG. 4A) and a conventionalalpha-amylase (FIG. 4B);

FIG. 5 is a graph showing GPG analysis (Sepharose CL-2B column) of thehydrolysis products of amylopectin incubated with purified SHE;

FIG. 6 is a graph showing GPG analysis (Sepharose CL-2B column) of thehydrolysis products of beta-limit dextrin incubated with purified SHE,overlaid on the graph of FIG. 5;

FIG. 7A is a graph showing GPC determination (Superose 6 column) of themolecular weight of SHE under native conditions;

FIG. 7B is a gel showing a molecular weight determination of SHE underdenaturing conditions by SDS-PAGE 7% analysis;

FIG. 8 is a chart showing the results of MALDI-TOF analysis of trypticpeptides produced from digestion of the 94 kD form of SHE;

FIGS. 9A and 9B show the nucleotide sequence of ZmAmy3 cDNA (SEQ IDNO: 1) aligned with the amino acid sequence (single letter code) of theencoded SHE protein (SEQ ID NO: 2), according to the invention;

FIGS. 10A and 10B show a sequence alignment of the SHE protein accordingto the invention (SEQ ID NO: 2) with the predicted polypeptide sequencesfor both the rice (SEQ ID NO: 15) and the Arabidopsis 100 kDalpha-amylases (AMY3) (SEQ ID NO: 16); and

FIG. 11 shows a hypothetical reaction mechanism for SHE activity.

DETAILED DESCRIPTION OF THE INVENTION

Two-dimensional native PAGE/activity gel analysis (i.e., starch zymogramanalysis) of proteins from developing maize kernels harvested 20 daysafter pollination (DAP) was used to identify a novel maize starchhydrolytic activity. As shown in FIG. 1, proteins in specific anionexchange chromatography fractions were separated by electrophoresisthrough a native polyacrylamide gel and then transferred to anotherpolyacrylamide gel containing starch. Enzymatic activity that alteredthe starch substrate in the gel was visualized by staining with iodinesolution. A distinct white activity band indicating starch hydrolysis,and not correlated with the activity of known starch hydrolytic enzymes,was identified in chromatography fractions 19-23.

This newly identified enzymatic activity was specifically isolated in aseries of purification steps, including ammonium sulfate precipitation,anion exchange chromatography, gel permeation chromatography andaffinity electrophoresis, as diagramed in FIG. 2. These purificationsteps resulted in a preparation of the enzyme that was devoid of otherstarch metabolizing activities. Those fractions containing purified SHEactivity were pooled and used to carry out various incubationexperiments with different polysaccharide substrates.

The glucan substrate specificity of this novel enzyme, called hereStarch Hydrolytic Enzyme (SHE), was determined by zymogram analysis. Asshown in FIG. 3, the specificity of SHE is similar, in general terms, tothat of previously known alpha-amylases. lBoth activities are able tohydrolyze starch (Sta), amylopectin (Ap), amylose (Am) and thebeta-limit dextrin of amylopectin (β-LD), but they are not able tohydrolyze the branched isomaltotriose polymer called pullulan (Pul).

Differences between SHE activity and that of conventional alpha-amylaseswere detected, however, after extended incubation of the purified SHEprotein with starch, amylopectin, and beta-limit dextrin. Eachindividual substrate was incubated overnight with SHE or with a lowermolecular weight alpha-amylase similarly purified from the same maizeendosperm tissue (a “conventional” alpha-amylase). The hydrolysisproducts were analyzed by fluorophore-assisted capillary electrophoresis(FACE) [7, 25]. As indicated in FIG. 4A, SHE activity does not releasesmall oligosaccharides (i.e., short chains consisting of 1 to 8 units ofglucose), in contrast to the activity of the conventional maizealpha-amylase (FIG. 4B).

The hydrolysis products were further characterized by gel permeationchromatography on a Sepharose CL-2B column. FIG. 5 displays the analysisof hydrolysis products that resulted from the incubation of amylopectinwith purified SHE. In addition, FIG. 6 compares the analysis of FIG. 5with that of hydrolysis products resulting from the incubation ofbeta-limit dextrin with SHE. These experiments demonstrate that purifiedSHE activity hydrolyzes both branched polysaccharides, as indicated bysignificant decreases in the molecular mass of each. However, theresults also indicate that the hydrolysis products themselves are ofhigh molecular weight, because no short glucosyl chains were detected bythe FACE analysis (see FIG. 4).

The apparent molecular weight of SHE was determined by gel permeationchromatography following passage of the purified protein over aSuperose-6 column. Comparison of the migration of the activity to thatof known molecular weight standards provided the estimate that SHE isapproximately 366 kD (FIG. 7A). However, analysis of purified SHE underdenaturing conditions by SDS-PAGE, followed by staining withCoomassie-Blue (FIG. 7B), revealed that the molecular weight of the SHEpolypeptide monomer is approximately 94 kD. This suggests that thepurified SHE activity results from the formation of an enzyme complexthat most likely is comprised of four SHE subunits.

The identity of the purified protein shown in FIG. 7B was established bysubjecting the 94 kD polypeptide to mass spectrometric analysis(MALDI-TOF) following trypsin digestion. The mass of each trypticpeptide was determined, and these were compared to the masses of trypticpeptides of all known proteins available in the databases. Databaseanalysis determined that the protein identity is closest to that of the100 kD alpha-amylase that is the predicted product of the rice largealpha-amylase gene (AMY3)(Genbank AP003408) (FIG. 8).

Based on the results of the mass spectrophotometric analysis, the riceAmy3 gene sequence was used to search the Maize Gene Database forsimilar sequences. This database contains a partial sequence of themaize genome, including “expressed sequence tags” (ESTs) representingpartial gene sequences. The database search uncovered a partial maizepolypeptide sequence predicted from a 548 nt EST sequence (GenbankPCO139185) that closely matches the predicted polypeptide sequences forboth the rice and the Arabidopsis 100 kD alpha-amylases (Atlg69830), asshown in FIG. 10.

The full-length coding sequence for the maize Amy3 (ZmAmy3) cDNA was PCRamplified using gene-specific primers complementary to the 3′ end of themaize EST and degenerate primers based on the 5′ region of the rice Amy3gene sequence. The 2640 bp ZmAmy3 cDNA product (FIGS. 9A and 9B, SEQ IDNO.:1) is predicted to code for a polypeptide of approximately 99 kDa(SHE) (FIGS. 9A and 9B, SEQ ID NO.:2). At the amino acid level, themaize and rice sequences are 97% identical over the length of thepolypeptide fragment predicted from the maize EST, and at the nucleicacid level the maize and rice sequences are 86% identical for thisregion. This high degree of sequence identity indicates that the maizeEST derives from the maize Amy3 gene and that the purified SHE proteinand the predicted rice AMY3 protein are homologous.

Comparisons of the deduced, full-length SHE amino acid sequence with thecorresponding AMY3 sequences from rice and Arabidopsis revealed that allthree large alpha-amylase polypeptides are closely conserved. Overall,the maize SHE sequence has 79% identity with the rice AMY3 polypeptideand 62% identity with the Arabidopsis AMY3 polypeptide. The rice andArabidopsis AMY3 amino acid sequences are 59% identical. In theN-terminal regions, the rice and maize AMY3 polypeptides are 60%identical. Because the three known AMY3 polypeptides (including maizeSHE) also have high sequence similarity to 50-60 kD AMY1 and AMY2sequences from rice, Arabidopsis, and maize at their C-termini, theyrepresent divergent plant alpha-amylase isoforms. The enzymatic activityof this class of alpha-amylase isoforms (AMY3) has not beencharacterized to date.

Use

The starch hydrolytic activity from maize kernels (SHE) described hereinexhibits a novel alpha-amylase activity. As indicated above, theactivity of this enzyme toward amylopectin results in hydrolysisproducts that are of the same approximate molecular mass as beta-limitdextrins. However, SHE activity does not result in the production ofmaltose or malto-oligosaccharides, as would be expected from any known,conventional alpha-amylase. This unique activity suggests that SHE is anendo-hydrolytic enzyme that specifically cleaves long amylopectin chains(B₂ or B₃ chains) that extend between unit clusters in the molecule,thus generating larger sized alpha-limit dextrins.

Referring to FIG. 11, a typical branched glucan substrate such asamylopectin 10, in which branch chains are arranged in distinct clusters12, 14 connected by single (non-branched) B₂ or B₃ chains 16, would beacted upon differently by SHE and a conventional alpha-amylase.According to this model, SHE, by virtue of its assembly state and size(a 366 kD tetramer 18), would be barred from access (pathway 20) to theglucan chains in the interior regions of amylopectin, clusters 12, 14.Thus, SHE hydrolysis would be limited only to those regions of theamylopectin molecule that are accessible to the enzyme, for example thelong linear chains 16 that extend between individual cluster units. Noother enzyme is known that cleaves starch specifically in regionsexternal to the unit clusters, producing larger sized alpha-limitdextrins 26. This is in stark contrast to the action (pathway 22) ofconventional, smaller sized alpha-amylases, which function as monomers24 and are likely to penetrate all regions of the amylopectin molecule,producing a mixture of small oligosaccharides and larger products 28.

Use of the ZmAmy3 cDNA that encodes SHE will make possible the isolationof active portions of SHE protein and, thus, the development of highlyactive, recombinant enzyme preparations for starch processing. Inaddition, ZmAmy3 cDNA can be used to isolate the cDNA encoding thehomologous AMY3 enzymes from other plants with important starchhydrolytic pathways, such as rice, Arabidopsis, apple and potato.

As indicated above, recombinant SHE and/or the native maize enzyme,recombinant AMY3 enzymes from other plant species and active fragmentsthereof will have value in starch processing for consistently generatingdifferent, larger sized alpha-limit dextrins (maltodextrins) forindustrial use. In comparison, the previously known alpha-amylases couldbe used to generate larger sized alpha-limit dextrins only bymanipulating enzyme concentrations and/or incubation times, reactionconditions that could not be counted on to consistently produce the sameproducts from batch to batch.

These new maltodextrin products will have value in food production asthickeners, emulsifiers, ice crystal retardants, texturizing agents,and/or fat or oil substitutes [13]. In manufacturing and pharmaceuticalindustries, the new maltodextrin products of SHE hydrolysis will havevalue as coating or encapsulation agents (e.g., for tablets or drugdelivery), or as adhesive or binding agents.

The new enzyme according to the invention, SHE, and its homologousequivalents also will have value for the production of high MW dextrinsthat can potentially be used for the manufacture of biopolymers. Nativeand destructured starches have long been employed as particulate fillersand in commodity plastics [33]. Biopolymer blends containing eitherchemically modified or native starch forms are continually beingexamined for their effectiveness as packaging materials and biomedicaladhesive agents [8, 22]. Large maltodextrins, such as those produced bySHE, may confer altered tensile properties and reduced water sensitivityto biopolymer blends.

Furthermore, the new enzyme according to the invention, SHE, and itshomologous equivalents will have value as unique enzymes that can beadded to formulations designed to selectively degrade starch. Inaddition, modification of the expression of the SHE enzyme and/or itshomologous forms in transgenic maize plants or in other transgenicorganisms (including bacteria, yeast, and other plant species) can beuseful for the generation of novel starch forms or altered starchmetabolism.

EXPERIMENTAL PROCEDURES Maize Stocks and Allele Nomenclature

Wild type maize inbred lines in the W64A or the Oh43 inbred geneticbackgrounds are used for analysis. Kernels are harvested 19-21 daysafter pollination (DAP), quick frozen in liquid nitrogen, and stored at−80° C. Prior to protein extraction, endosperm tissue is separated fromembryo and pericarp tissues.

The nomenclature follows the standard maize (Zea mays L.) geneticsformat [1]. Names and symbols of genetic loci are italicized. MessengerRNAs and cDNAs are designated by italic font with the first lettercapitalized, whereas polypeptide symbols are not italicized and are inupper case letters. Species designations for orthologous loci aredistinguished by having the first letter of both the genus and thespecies precede the locus designation (e.g., ZmAmy3 designates the Zeamays Amy3 cDNA).

Protein Extraction and Activity Gel Analysis

Protein isolation from endosperm is as described [7]. Briefly, frozenkernels (5 g) are ground to a fine powder in liquid nitrogen with amortar and pestle, and the tissue is suspended in 5 mL of buffercontaining 50 mM sodium acetate, pH 6, and 20 mM DTT. All of the lysatesare centrifuged at 50,000 g for one hour at 4° C. Protein concentrationsare determined according to the method of Bradford [3, 4].

For one-dimensional native PAGE activity gel analysis (i.e., zymogramanalysis), total proteins (approximately 100 μg) are separated on anative polyacrylamide gel (16 cm×20 cm×0.15 cm). The resolving gelcontains 7% (w/v) acrylamide (29:1 acrylamide-bisacrylamide [Sigma]) and375 mM Tris-HCl, pH 8.8. The stacking gel contains 4% (w/v) acrylamideand 63 mM Tris-HCl, pH 6.8. Electrophoresis is conducted at 4° C., 25 Vcm⁻¹ for 4 h using a Protean II cell (Bio-Rad) in an electrode buffer of25 mM Tris, 192 mM glycine, pH 8.8, and 2 mM DTT. At the end of the run,the gel is electroblotted to a polacrylamide gel of the same sizecontaining 7% acrylamide, 0.3% (w/v) potato starch (Sigma), and 375 mMTris-HCl, pH 8.8. Alternative substrates to starch in the transfer gelinclude 0.3% (w/v) amylopectin (Sigma), 0.3% (w/v) amylose (Sigma), 0.3%(w/v) beta limit-dextrin (Megazyme), 0.3% (w/v) oyster glycogen (Sigma),and 0.3% (w/v) azure pullulan (Sigma). The transfer is performedovernight at 20 V in the electrode buffer at room temperature. Starchmetabolic activities are observed by staining the gel with I₂/KIsolution, and the gel is photographed immediately.

For two-dimensional zymogram analysis, total proteins (40 μg) areextracted as described above and loaded onto an anion exchangechromatography (MonoQ HR 5/5) using AKTA FPLC instrumentation(Amersham-Pharmacia). The MonoQ column is equilibrated with buffer A (50mM Tris-acetate, pH 7.5; 10 mM DTT). Bound proteins are eluted with a 48mL-linear gradient of 0 to 500 mM NaCl in buffer A containing 1M NaCl.The flow rate is 0.9 mL/min, and 1 mL fractions are collected. Proteinsin each fraction are separated by non-denaturing PAGE. Followingelectrophoresis, proteins are transferred by electroblotting to apolyacrylamide gel of the same size containing 0.36 (w/v) starch. Starchmetabolic activities are observed after staining the gel with I₂/KIsolution, as described [7]. Transfer is performed overnight as describedfor the one-dimensional zymogram.

Protein Purification

The unknown glucan hydrolytic activity (termed “SHE”) detected by one-and two-dimensional native PAGE activity gel analysis was purified in astep-wise manner from crude protein extracts isolated from approximately100 grams mid-development maize kernels. At the conclusion of eachpurification step, starch zymogram analysis was employed to identify thefraction(s) containing SHE activity. The first step in the purificationscheme was fractionation by ammonium sulfate precipitation, in whichproteins were precipitated by the slow addition of saturated ammoniumsulfate to 40% saturation. After incubation at 0° C. for 30 min,proteins were collected by centrifugation at 20,000 g for 20 min. Theprotein pellet was dissolved in 5 mL Buffer A and dialyzed twice against400 mL Buffer A, according to previously described methods [5].

Dialyzed proteins were injected onto a FPLC MonoQ HR 5/5 column usingAKTA FPLC instrumentation (Amersham-Pharmacia), preincubated with BufferA. Bound proteins were eluted with a 48 mL-linear gradient of 0 to 500mM NaCl in buffer A containing 1M NaCl. The flow rate was 0.9 mL/min,and 1 mL fractions were collected. MonoQ fractions containing SHEactivity were pooled and further purified by gel filtrationchromatography (GPC) on a Sephacryl S400 column (Amersham-Pharmacia).GPC was performed at 4° C. in buffer A at a flow rate of 0.4 mL/min, and1 mL fractions were collected. Fractions containing SHE activity werepooled and concentrated using an Amicon centricon microspin column(Millipore) to 500 μL. A second GPC purification was performed byapplication of 100 μL of the concentrated SHE-containing sample to aSuperose 6 column (Amersham-Pharmacia). Superose 6 GPC was at 4° C. inbuffer A with a flow rate of 0.2 mL/min, and 0.3 mL fractions werecollected. Identification of the Superose 6 fractions containing SHEactivity was achieved by starch zymogram analysis, which also providedthe final step in the purification process, determination of theaffinity of the purified protein for the starch substrate in the gel.

Purification of “conventional” alpha amylase from maize was achieved bythe same methods as described for the purification of SHE. In this case,the alpha amylase activity was monitored at each step in thepurification process enzymatically, using the Ceralpha kit (Megazyme).Briefly, this method assays the production of p-nitrophenol at 410 nm,which results from the hydrolysis of non-reducing ends that are blockedwith p-nitrophenyl maltoheptaoside. Assays for “conventional” alphaamylase activity were conducted at 30° C. for 30 min and were terminatedby the addition of 1% (w/v) Trizma base (Sigma).

Characterization of Purified Protein

The approximate molecular mass of SHE was determined by comparison ofthe elution of SHE from the analytical Superose 6 column to the elutionof known MW standards from same column. Standard proteins used tocalibrate the column were bovine thyroglobulin (670,000), bovine gammaglobulin (158,000), chicken ovalbumin (44,000), horse myoglobin (17,000)and vitamin B-12 (1,350) (Bio-Rad).

Protein in pooled, concentrated Superose 6 fractions containing SHEactivity was analyzed by SDS-PAGE on a 7% polyacrylamide gel, followedby staining of the gel with Coomassie brilliant blue and destaining with50% methanol solution, according to standard procedures [29]. Theapproximate molecular mass of the strongly stained, abundant polypeptidecorresponding to SHE was determined by comparison of the migrationdistance of the polypeptide with the migration of commercial molecularweight standards (Bio-Rad).

To identify the SHE protein in terms of its amino acid sequence, theband corresponding to SHE was excised from the SDS-polyacrylamide geland analyzed by time-of-flight mass spectrometry (MALDI-TOF) accordingto standard methods [21]. At the mass spectrometry facility (ProteinFacility, Iowa State University), the polypeptide was digested with theprotease trypsin. The peptide fragments were concentrated, fractionatedby capillary electrophoresis, and the eluent from the capillary wasdirectly injected into the electrospray mass spectrometer, whichseparated the individual peptides. Computational algorithms utilized thedifferences between fragment masses to reveal the amino acid sequence ofthe original peptide, based on the expectation of fragmentation bycleavage of the peptide bonds. The peptide sequences were then comparedto proteins in the public databases, enabling the sequence match of apeptide in a given gel band to a peptide within a protein sequence inthe database.

Characterization of Hydrolysis Products

Purified SHE activity was analyzed by incubation of 20 μL of the pooledand concentrated Superose 6 fractions containing SHE activity with 100μL of a 1% amylopectin (Sigma) or 1% beta limit-dextrin (Megazyme)solution. in a total volume of 200 μL. Incubation was at 37° C. for 24h. Control incubations with both substrates also were conducted using“conventional” maize alpha amylase purified from developing maizeendosperm, under the same conditions described for SHE incubation.Equivalent amounts of SHE and the alpha amylase control proteins weredetermined by quantification of the protein sample according to standardmethods [3].

SHE amylopectin hydrolysis products were analyzed using a modifiedprotocol for fluorophore-assisted carbohydrate electrophoresis (FACE)[7, 25]. Briefly, a 10 μL aliquot of the SHE amylopectin hydrolysisproducts was lyophilized, then resuspended in 30% DMSO and boiled for 10min. A 10 μL aliquot was diluted to a final volume of 50 μL with 50 mMsodium acetate, pH 4.5. Pseudomonas sp. isoamylase (1 μL, 0.3 units)(Catalog No. E-ISAMY, Megazyme International, Bray, Ireland) was addedand the reaction incubated overnight at 42° C. The mixture was heated inboiling water for 5 min and then centrifuged for 2 min at 12,000 g. A 10μL sample of the reaction was evaporated to dryness in a Speed Vac. Thereducing ends of the liberated oligosaccharide chains were derivatizedwith the fluorescent compound 8-amino-1,3,6-pyrenetrisulfonic acid(APTS) (Catalog No. 09341, Sigma-Aldrich, St. Louis, Mo.) by suspendingthe dried sample in 2 μL of 1 M sodium cyanoborohydride intetrahydrafuran (Catalog No. 29,681-3, Sigma-Aldrich) and 2 μL APTS (0.1mg/μL in 15% acetic acid). The reaction was incubated overnight at 42°C., diluted with 46 μL water, vortexed, and centrifuged briefly in amicrofuge. A 5 μL aliquot was added to 195 μL purified water, and thissample was applied to a Beckman P/ACE capillary electrophoresisinstrument. The sample injection parameters were 5 s at 0.5 psi.Separation was accomplished at 23.5 kV in an uncoated capillary usingCarbohydrate Separation Gel Buffer N (Catalog Nos. 338451 and 477623,respectively, Beckman Coulter, Inc., Fullerton, Calif.).

SHE amylopectin and beta-limit dextrin hydrolysis products also wereanalyzed by GPC, using a Sephacryl CL-2B column (Amersham-Pharmacia;0=18 cm; H=50 cm) equilibrated with 10 mM NaOH. A 100 μL volume of thehydrolysis product was applied to the CL-2B column, and eluted at a flowrate of 12 mL/h in 1.4 mL fractions. Aliquots (30 μL) of each fractionwere incubated with 50 μL amyloglucosidase solution (0.3 U in a 50 mMsodium citrate buffer, pH 4.6; Megazyme). The μg of glucose equivalentsin each fraction was determined by the colorimetric glucoseoxidase/peroxidase method (Sigma Diagnostics).

PCR Amplification, Cloning of ZmAmy3 cDNA and Nucleotide SequenceAnalysis of the ZmAmy3 cDNA

Total RNA was isolated from approximately 10 g Zea mays kernels (Oh43inbred background) harvested 19 days after pollination, using amodification of the protocol reported by Chomczynski and Sacchi [6].Briefly, frozen kernels were ground to a powder in liquid nitrogen, andRNA was extracted with Trizol reagent (Invitrogen). Addition ofchloroform separated polysaccharides and DNA from the RNA-containingaqueous fraction. The RNA was then precipitated and air-dried,resuspended in water, and treated with DNase. The RNA was furtherpurified using an RNeasy Plant Mini Kit (Qiagen).

Approximately 5 μg total RNA from developing maize kernels was reversetranscribed (RT) using a Superscript III First-Strand Synthesis Systemfor RT-PCR kit (Invitrogen), using the oligo-(dT)18 primer provided. TheRT product was used as the template for PCR amplification of a 2451 bpfragment of the ZmAmy3 cDNA. Five ZmAmy3 gene-specific primers weredesigned based on the sequence of an EST fragment in the maize genome(Genbank accession number PCO139185). These primers are designated KS052(5′-GCC AAG TCT ATG AAG ACG CTT CC-3′) (SEQ ID NO: 3), KS055 (5′-GCT GATGGA GCA GGA AAC TC-3′) (SEQ ID NO: 4), KS056 (5′-CTT CAG GCG ACA CAG AATCA-3′) (SEQ ID NO: 5), KS057 (5′-CTA CAA TCA GGA TGC CCA CA-3′) (SEQ IDNO: 6), and KS058 (5′-AAC AAA GTT GAC AGC GGC GAT TGG A-3′) (SEQ ID NO:7). Degenerate primers were designed based on predicted orthologoussequences of the Arabidopsis thaliana Amy3 gene (Genbank accessionnumber BT000643) and Oryza sativa Amy3 gene (Genbank accession numberNM_(—)191752). The degenerate primers are KS047 (5′-GGV AAR TGG GTS TTRCAT TGG GG-3′) (SEQ ID NO: 8) and KS048 (5′-GGV AAR TGG GTS CTS CAT TGGGG-3′) (SEQ ID NO: 9) (V=A, C or G; R=A or G; S=G or C). PCRamplification was conducted according to the protocol specified by theAccuzyme Pfx PCR Amplification kit (Invitrogen), using 500 μg of thetemplate DNA, and equivalent amounts (0.5 μmol, final concentration) ofprimers KS052, KS047, and KS048. The complete nucleotide sequence ofboth strands of the amplified fragment was obtained using the three PCRprimers as well as primers KS055, KS056, and KS057.

To obtain the 5′ end of the ZmAmy3 cDNA, the rapid amplification of cDNAends (RACE) [10] protocol was employed, using the GeneRacer kit(Invitrogen), according to instructions provided with the kit. Briefly,5 μg RNA from maize kernels was reverse transcribed using theZmAmy3-specific primer KS052 (0.5 μmol, final concentration). PCRamplification of the 5′ end was performed with Accuzyme Pfx polymerase,according to the kit protocol, using primer KS052 and a primer providedby the GeneRacer kit (5′-CGA CTG GAG CAC GAG GAC ACT GA-3′) (SEQ ID NO:10). The amplified DNA fragments were gel purified using the QiaquickGel Extraction kit (Qiagen). The purified PCR product was used as thetemplate for a second-round PCR reaction using a nested ZmAmy3-specificprimer KS059 (5′-GGG CTG TCC TTC TGA ATT GGG CAA A-3′) (SEQ ID NO: 11)and a nested GeneRacer primer (5′-GGA CAC TGA CAT GGA CTG AAG GAG TA-3′)(SEQ ID NO: 12). The amplified products were gel purified and sequencedusing the same primers that were used for the amplification.

The PCR fragment containing the amplified ZmAmy3 cDNA was re-amplifiedfor the purpose of cloning the fragment into a plasmid vector. Followinga protocol based on the recombination-mediated cloning strategy of theGateway Technology system (Invitrogen), two new PCR primers were usedfor the amplification: KS062 (5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTGGGA AGT GGG TAC TGC ACT GGG G-3′) (SEQ ID NO: 13), and KS063 (5′-GGG GACCAC TTT GTA CAA GAA AGC TGG GTG CCA AGT CTA TGA AGA CGC TTC C-3′) (SEQID NO: 14). The PCR product was gel purified and cloned into the Gatewaycloning vector pDONR221 using the Invitrogen BP Clonase Enzyme Mix,according to the recommended protocol, generating plasmid pKS024. E.coli cells (DH-5α) were transformed with the plasmid DNA and screenedfor successful transformation events on LB media containing kanamycin.Plasmid DNA was isolated from successful transformants and the identityof pKS024 is confirmed by restriction enzyme analysis. Plasmid DNA wasdigested with both ApaI and HindIII, which produces fragments of 3738and 1114 bp; BamHI, which produces fragments of 4449 and 553 bp; andwith EcoRV and produces fragments of 2903 and 2100 bp.

The nucleotide sequence of plasmid pKS024 was determined by the chaintermination method [30] using Sequenase Version 2.0 (U.S. BiochemicalCorp.). The plasmid has been deposited with the American Type CultureCollection.

Deposits

Plasmid pKS024 was deposited on Sep. 24, 2004, with the American TypeCulture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 USA, asATCC No. PTA-6235.

Applicants' assignee, Iowa State University Research Foundation,represents that the ATCC is a depository affording permanence of thedeposit and ready accessibility thereto by the public if a patent isgranted. All restrictions on the availability to the public of thematerial so deposited will be irrevocably removed upon the granting of apatent. The material will be available during the pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and 35 USC 122. The deposited material will bemaintained with all the care necessary to keep it viable anduncontaminated for a period of at least five years after the most recentrequest for the furnishing of a sample of the deposited microorganism,and in any case, for a period of at least thirty (30) years after thedate of deposit or for the enforceable life of the patent, whicheverperiod is longer. Applicants' assignee acknowledges its duty to replacethe deposit should the depository be unable to furnish a sample whenrequested due to the condition of the deposit.

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While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

1. A nucleic acid isolate comprising a nucleotide sequence encoding thealpha-amylase having the amino acid sequence of SEQ ID NO:2.
 2. Anucleic acid isolate comprising the complement of a nucleotide sequenceencoding the alpha-amylase having the amino acid sequence of SEQ IDNO:2.
 3. A nucleic acid isolate comprising the nucleotide sequence ofSEQ ID NO:1.
 4. A nucleic acid isolate having the nucleotide sequence ofSEQ ID NO:1.
 5. A nucleic acid isolate comprising the complement of thenucleotide sequence of SEQ ID NO:1.
 6. A nucleic acid isolate having thenucleotide sequence of the complement of SEQ ID NO:1.
 7. A transgenicplant comprising a genome including a foreign DNA sequence comprising asequence encoding the alpha-amylase having the amino acid sequence ofSEQ ID NO:2.
 8. A transgenic plant comprising a genome including aforeign DNA sequence encoding the alpha-amylase having the amino acidsequence of SEQ ID NO:2.
 9. A transgenic plant comprising a genomeincluding a foreign DNA sequence comprising the complement of a sequenceencoding the alpha-amylase having the amino acid sequence of SEQ IDNO:2.
 10. A transgenic plant comprising a genome including, as a foreignDNA sequence, the complement of the DNA sequence encoding thealpha-amylase having the amino acid sequence SEQ ID NO:2.
 11. Arecombinant expression vector comprising the nucleic acid isolate ofclaim
 1. 12. A composition comprising a cell transformed with therecombinant expression vector of claim
 11. 13. A recombinant expressionvector comprising the nucleic acid isolate of claim
 2. 14. A compositioncomprising a cell transformed with the recombinant expression vector ofclaim 13.